Crystalline forms of an 8-Azabicyclo(3.2.1)octane compound

ABSTRACT

The invention provides a crystalline sulfate salt of 3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide or a solvate thereof. The invention also provides pharmaceutical compositions comprising such crystalline salt forms, methods of using such crystalline salt forms to treat diseases associated with mu opioid receptor activity, and processes useful for preparing such crystalline salt forms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/904,090, filed on Feb. 28, 2007, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to crystalline salt forms of an8-azabicyclo[3.2.1]octane compound which are useful as mu opioidreceptor antagonists. The invention is also directed to pharmaceuticalcompositions comprising such crystalline compounds, methods of usingsuch compounds for treating or ameliorating medical conditions mediatedby mu opioid receptor activity, and processes useful for preparing suchcompounds.

2. State of the Art

Commonly-assigned U.S. Provisional Application Nos. 60/777,962, filed onMar. 1, 2006, and 60/841,028, filed on Aug. 30, 2006, and U.S.application Ser. No. 11/711,961 disclose 8-azabicyclo[3.2.1]octanecompounds that are mu opioid receptor antagonists that are expected tobe useful for treating or ameliorating medical conditions mediated by muopioid receptor activity. In particular, the compound3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate is specifically disclosed in these applications as demonstratingmu opioid receptor antagonist activity.

The chemical structure of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide(hereinafter compound 1) is shown below:

To effectively use this compound as a therapeutic agent, it would bedesirable to have a solid-state salt form that can be readilymanufactured and that has acceptable chemical and physical stability.For example, it would be highly desirable to have a salt form that isthermally stable, for example at temperatures exceeding about 175° C. orabout 180° C., and is not hygroscopic nor deliquescent, therebyfacilitating processing and storage of the material. Crystalline solidsare generally preferred over amorphous forms, for enhancing purity andstability of the manufactured product.

No crystalline salt forms of compound 1 have previously been reported.Accordingly, a need exists for a stable, crystalline salt form ofcompound 1 that is neither hygroscopic nor deliquescent, and exhibitsfavorable thermal stability.

SUMMARY OF THE INVENTION

The present invention provides a crystalline sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideor a solvate thereof. In one aspect, the crystalline salt form of theinvention is a crystalline sulfate salt of compound 1. In anotheraspect, the crystalline salt form of the invention is a crystallinehydrate of the sulfate salt of compound 1.

Surprisingly, the crystalline sulfate salt of the invention has beenfound to exhibit no significant thermal events below a meltingtemperature in the range of about 190 to about 205° C. and to exhibit aweight change of less than about 0.3% when exposed to a range ofrelative humidity between about 2% and about 90% at room temperature.Furthermore, neither the crystalline sulfate salt of the invention northe hydrate thereof is deliquescent when exposed to up to about 90%relative humidity at room temperature.

Among other uses, the crystalline salt forms of the invention areexpected to be useful for preparing pharmaceutical compositions fortreating or ameliorating medical conditions mediated by mu opioidreceptor activity. Accordingly, in another of its composition aspects,the invention provides a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a crystalline sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideor a solvate thereof.

The invention also provides a method of treating or ameliorating adisease or condition ameliorated by treatment with a mu opioid receptorantagonist, e.g. a disorder of reduced motility of the gastrointestinaltract, the method comprising administering to the mammal, atherapeutically effective amount of a crystalline sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideor a solvate thereof.

The invention further provides a method of treating opioid-induced boweldysfunction or post-operative ileus, the method comprising administeringto the mammal, a therapeutically effective amount of a crystallinesulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideor a solvate thereof.

In another method aspect, the invention provides a process for preparinga crystalline sulfate salt of the invention, the process comprisingcontacting3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidewith sulfuric acid to form a reaction mixture, and isolating thecrystalline sulfate salt from the reaction mixture.

The invention provides an additional process for preparing a crystallinesulfate salt of the invention, the process comprising dispersing acrystalline hydrate of the sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidein a diluent comprising methanol to form a reaction mixture, andisolating the crystalline sulfate salt from the reaction mixture In yetanother method aspect, the invention provides a process for preparing acrystalline sulfate salt of compound 1, the process comprising: (a)contacting a protected precursor to compound 1 in which the hydroxygroups are protected, with sulfuric acid to form a first reactionmixture; (b) isolating an intermediate grade solid sulfate salt ofcompound 1 from the first reaction mixture; (c) dispersing theintermediate grade solid sulfate salt in a diluent comprising methanolto form a second reaction mixture; and (d) isolating the crystallinesulfate salt from the second reaction mixture.

In a related composition aspect, the invention provides theN-cyclohexylmethyl-(2-oxoethyl)-carbamic acid benzyl ester bisulfiteadduct, which is useful for the preparation of the above protectedprecursor to compound 1.

The invention also provides a crystalline sulfate salt of the inventionas described herein for use in therapy or as a medicament, as well asthe use of a crystalline sulfate salt of the invention in themanufacture of a medicament, especially for the manufacture of amedicament for treating a disorder associated with mu opioid receptoractivity in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows an x-ray powder diffraction (XRPD) pattern of a crystallinesulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideof the invention.

FIG. 2 shows a differential scanning calorimetry (DSC) trace (right sidevertical axis) and a thermal gravimetric analysis (TGA) trace (left sidevertical axis) for a crystalline sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideof the invention.

FIG. 3 shows a dynamic moisture sorption (DMS) trace for a crystallinesulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideof the invention.

FIG. 4 shows an x-ray powder diffraction (XRPD) pattern of a crystallinehydrate of a sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideof the invention.

FIG. 5 shows a differential scanning calorimetry (DSC) trace (right sidevertical axis) and a thermal gravimetric analysis (TGA) trace (left sidevertical axis) for a crystalline hydrate of a sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideof the invention.

FIG. 6 shows a dynamic moisture sorption (DMS) trace for a crystallinehydrate of a sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideof the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a crystalline sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideor a solvate thereof.

DEFINITIONS

When describing the compounds, compositions and methods of theinvention, the following terms have the following meanings, unlessotherwise indicated.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition in a patient, such as a mammal(particularly a human) which includes:

-   -   (a) preventing the disease, disorder, or medical condition from        occurring, i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease, disorder, or medical condition,        i.e., eliminating or causing regression of the disease,        disorder, or medical condition in a patient, including        counteracting the effects of other therapeutic agents;    -   (c) suppressing the disease, disorder, or medical condition,        i.e., slowing or arresting the development of the disease,        disorder, or medical condition in a patient; or    -   (d) alleviating the symptoms of the disease, disorder, or        medical condition in a patient.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of the invention or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is specifically termed a hydrate.

The term “crystalline sulfate salt”, or alternatively “crystallinesulfate salt (anhydrous form)” or “anhydrous sulfate salt”, as usedherein means a crystalline solid that does not include a substantiallyfixed molar fraction of solvent molecules in the crystal lattice, i.e.one that is not a solvate or hydrate. Solvates, or specificallyhydrates, of the invention are identified explicitly.

It must be noted that, as used in the specification and appended claims,the singular forms “a”, “an”, “one”, and “the” may include pluralreferences, unless the content clearly dictates otherwise.

Active Agent

The active agent in the present salt forms, i.e. compound 1, isdesignated3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide.Alternatively, using the IUPAC conventions as implemented in AutoNomsoftware, (MDL Information Systems, GmbH, Frankfurt, Germany), thecompound is denoted3-((1R,3R,5S)-8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-azabicyclo[3.2.1]oct-3-yl)benzamide.The name used herein therefore corresponds to the IUPAC notation withthe endo orientation of the substituted phenyl group with respect to the8-azabicyclo[3.2.1]octane group indicated explicitly. In yet othercommon nomenclatures, the “((S)-2,3-dihydroxy-propionyl)amino” portionof the molecule is variously designated as((S)-2,3-dihydroxy-1-oxopropyl)amino or ((S)-2,3-dihydroxypropanamido)

Salt Forms of the Invention

In one aspect, the invention provides crystalline3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate.

In one aspect, the crystalline sulfate salt of the present invention ischaracterized by an x-ray powder diffraction (XRPD) pattern having twoor more diffraction peaks, including three or more and four or morediffraction peaks, at 2θ values selected from 6.58±0.20, 7.52±0.20,9.35±0.20, 14.69±0.20, 16.01±0.20, 17.45±0.20, 17.99±0.20, 18.62±0.20,19.76±0.20, 21.11±0.20, 22.07±0.20, 23.18±0.20, 23.74±0.20, 24.56±0.20,25.63±0.20, 26.45±0.20, 27.86±0.20, 28.31±0.20, 29.54±0.20, 30.59±0.20,31.58±0.20, 33.89±0.20, and 36.02±0.20. In particular, in this aspect,the crystalline form is characterized by an x-ray powder diffractionpattern having two or more diffraction peaks, including three or moreand four or more diffraction peaks, at 2θ values selected from14.69±0.20, 16.01±0.20, 21.11±0.20, 22.07±0.20, and 23.18±0.20.

As is well known in the field of powder x-ray diffraction, peakpositions of XRPD spectra are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, a crystallinesulfate salt of compound 1 is characterized by an x-ray powderdiffraction pattern in which the peak positions are substantially inaccordance with those shown in FIG. 1.

The crystalline sulfate structure has been further characterized bysingle crystal x-ray diffraction analysis, providing the followinglattice parameters: unit cell is orthorhombic with dimensions a=6.8239Å, b=16.2275 Å, c=24.2021 Å, α=β=γ=90°; cell volume (V) of 2680.0 Å³;calculated density is 1.38 g/cm³; space group is P2₁2₁2₁(#19). Theresulting molecular structure confirms the chemical composition is thatof the sulfate salt of compound 1 in a 1:1 molar ratio of sulfatecounterion to compound 1 and that the asymmetric unit cell does notcontain water or other solvent molecules. X-ray powder diffraction peakspredicted from the derived atomic positions are in excellent agreementwith the observed XRPD pattern.

In another aspect, the crystalline sulfate salt of the present inventionis characterized by its behavior when exposed to high temperature. Asdemonstrated in FIG. 2, the differential scanning calorimetry (DSC)trace of a highly crystalline sample exhibits a peak in endothermic heatflow, identified as a melt transition, in the range of about 190° C. toabout 205° C. The thermal gravimetric analysis (TGA) trace shows nosignificant weight loss at temperatures below the melting point. Thermaldecomposition occurs approximately upon melting.

In yet another aspect a crystalline sulfate salt is characterized by itsinfrared absorption spectrum which shows significant absorption bands atabout 430, 590, 639, 705, 867, 1036, 1053, 1105, 1171, 1231, 1277, 1375,1391, 1452, 1476, 1553, 1596, 1639, 1664, 2852, 2907, 2928, 2967, 3168,and 3357 cm⁻¹.

A crystalline sulfate salt of compound 1 has been demonstrated to have areversible sorption/desorption profile with an exceptionally low levelof hygroscopicity (i.e., less than about 0.3% weight gain in thehumidity range of 2% relative humidity to 90% relative humidity at roomtemperature) as shown in FIG. 3.

Additionally, the crystalline sulfate salt of compound 1 has been foundto be stable upon exposure to elevated temperature and humidity. Afterstorage for 4 weeks at 40° C. and 75% relative humidity, analysis byHPLC showed no chemical degradation and there were no detectable changesin the DSC, TGA, or XRPD results.

In another aspect, the invention provides a crystalline hydrate of asulfate salt of compound 1.

In one aspect, a crystalline hydrate of a sulfate salt of the presentinvention is characterized by an x-ray powder diffraction (XRPD) patternhaving two or more diffraction peaks, including three or more and fouror more diffraction peaks, at 2θ values selected from 9.41±0.20,9.98±0.20, 15.17±0.20, 16.70±0.20, 18.59±0.20, 19.46±0.20, 19.91±0.20,20.63±0.20, 21.35±0.20, 21.89±0.20, 23.00±0.20, 24.20±0.20, 25.40±0.20,26.03±0.20, 27.44±0.20, 28.46±0.20, 29.45±0.20, 31.22±0.20, 31.82±0.20,33.17±0.20, 33.56±0.20 and 36.89±0.20. In particular, in this aspect,the crystalline form is characterized by a x-ray powder diffractionpattern having two or more diffraction peaks, including three or moreand four or more diffraction peaks, at 2θ values selected from16.70±0.20, 18.59±0.20, 19.46±0.20, 19.91±0.20, 23.00±0.20, and24.20±0.20.

In another aspect, a crystalline hydrate of a sulfate salt of compound 1is characterized by an x-ray powder diffraction pattern in which thepeak positions are substantially in accordance with those shown in FIG.4.

The crystalline hydrate of a sulfate salt of the present invention isalso characterized by its differential scanning calorimetry (DSC) tracewhich exhibits two endothermic events: a first peak in endothermic heatflow in the range of about 125 to about 133° C. and a second peak in therange of about 178 to about 183° C. when analyzed at a heating rate of10° C. per minute as illustrated in FIG. 5. The thermal gravimetricanalysis (TGA) trace shows a first thermal event between about 60 andabout 140° C. and a second thermal event between about 140 and about190° C. Analysis by TGA coupled with IR of the material vaporized in thefirst thermal event is consistent with a hydrate composition havingabout one mole of water per mole of compound 1 sulfate.

Surprisingly, a crystalline hydrate of a sulfate salt of compound 1 hasdemonstrated low hygroscopicity. As illustrated in FIG. 6 thecrystalline hydrate exhibits a reversible sorption/desorption profile atroom temperature over the entire range of about 2% to about 90% relativehumidity with less than about 0.3% weight gain over the entire range inrelative humidity.

These properties of the salt forms of this invention are furtherillustrated in the Examples below.

Synthetic Procedures

The active agent,3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide,can be prepared from readily available starting materials using theprocedures described in the Examples below, or using the proceduresdescribed in the commonly-assigned U.S. applications listed in theBackground section of this application.

In one method of preparation, a crystalline sulfate salt of theinvention is prepared by contacting3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidewith about 0.5 to about 1.5 molar equivalents, including about 1 molarequivalent, of sulfuric acid. Generally, this reaction is conducted inan inert diluent at a temperature ranging from about 0° C. to about 65°C., including about 60 to about 65° C. Suitable inert diluents include,for example, methanol, toluene, dichloromethane, and combinations suchas toluene and acetonitrile, dichloromethane and acetonitrile, andtoluene, acetonitrile, and water, in addition to a methanol and watercombination comprising about 10% water. Using these diluents, a reactionmixture with a concentration of between about 5 and about 400 mg/mL,including between about 50 and about 100 mg/mL, is prepared and held forbetween about 2 and about 24 hours, with optional agitation. The mixturemay be cooled to between about 5 and about 20° C. during the holdingperiod.

Upon completion of the reaction, a crystalline salt of the invention isisolated from the reaction mixture by any conventional means, such asfiltration, concentration, centrifugation, and the like.

Alternatively, the crystalline sulfate of the invention is prepared byrecrystallization of the hydrate form. The crystalline hydrate isdispersed in an inert diluent as described above at a concentration ofbetween about 5 and about 400 mg/ml. Methanol, or a methanol:watercombination, in a ratio of from about 3:1 to about 9:1, are particularlyuseful diluents for this reaction. The reaction mixture is maintained attemperatures in the range of about 0 to about 65° C., typically withagitation, for between about 1 and about 24 hours, including betweenabout 1 and about 6 hours. Typically the reaction mixture is cooled fromabout 65° C. to between about 5 and about 20° C. during the holdingperiod. To improve the yield, the volume of the solution may be reducedby about 50% before holding the reaction mixture for a time period ofbetween about 1 and about 24 hours, including between about 1 and about6 hours, at a temperature of between about 5 and about 20° C. Theresulting crystals are recovered conventionally.

Both the crystalline sulfate salt and the crystalline hydrate of thesulfate salt of compound 1 are advantageously prepared from theprotected precursor of compound 1. As described in the examples below,to prepare the active agent, the protected aldehyde 2,N-cyclohexylmethyl-(2-oxoethyl)-carbamic acid benzyl ester, regeneratedfrom its bisulfite adduct 3, is coupled with3-endo-(8-azabicyclo[3.2.1]oct-3-yl)-benzamide hydrochloride 4 toprovide protected intermediate 5, which is deprotected to provide3-endo-{8-[2-(cyclohexylmethylamino)ethyl]-8-aza-bicyclo[3.2.1]oct-3-yl}benzamide6.

Reaction of intermediate 6 with lithium(4S)-2,2-dimethyl-1,3-dioxolane-4-carboxylate 7 provides the protectedintermediate (S)-2,2-dimethyl-[1,3]dioxolane-4-carboxylic acid{2-[3-(3-carbamoyl-phenyl)-8-azabicyclo[3.2.1]oct-8-yl]ethyl}cyclohexylmethyl-amide8

Protected intermediate 8 is contacted with between about 0.8 and about1.3 equivalents, typically about 1 to about 1.2 equivalents, of aqueoussulfuric acid in an inert diluent, such as ethyl acetate or isopropylacetate, at a temperature of between about 20 and about 30° C. A seconddiluent, which is miscible with the reaction mixture, and in which theproduct is less soluble, is typically included in the reaction mixture.Acetonitrile is useful as the second diluent. The reaction mixture istypically stirred for a period of between about 2 and about 72 hoursresulting in deprotection of compound 8 and formation of theintermediate grade solid sulfate salt of compound 1, which is typicallypredominantly the crystalline hydrate of the sulfate of compound 1. Theintermediate grade product can be isolated conventionally, for exampleby filtration.

The hydrate form may be obtained by recrystallization of theintermediate grade sulfate product, for example, by suspending theintermediate grade product in acetonitrile with heating, adding water topromote dissolution, cooling to ambient temperature, and isolating therecrystallized hydrate form, as described in Example 2 below.

The crystalline sulfate salt of compound 1 may be obtained from theintermediate grade solid product of the deprotection step describedabove. The intermediate grade product is dispersed in an inert diluentcomprising methanol at a concentration between about 5 and about 400mg/mL, including between about 50 and about 200 mg/mL. Surprisingly, ithas been determined that a methanol and water combination, having up to25% water, including between about 0 and about 15% water, and betweenabout 5 and about 15% water, is a useful diluent for preparing ananhydrous crystalline salt. In particular, a methanol and watercombination comprising about 10% water is useful for recrystallizationof the intermediate grade product to the anhydrous sulfate salt of thepresent invention.

In a typical recrystallization process, the reaction mixture is heateduntil complete dissolution is obtained, for example the reaction mixtureis heated to about 65° C., and then cooled to between about 5 and about22° C. over a period of between about 2 and about 24 hours. Optionally,seed crystals of the anhydrous sulfate salt can be added when thereaction mixture is below the dissolution temperature. The resultingcrystals are recovered conventionally, for example, by filtration.

According to yet another process, the hydrate form may be prepared fromthe crystalline sulfate (anhydrous) form. Typically, the crystallinesulfate is first converted to a more soluble, amorphous form, forexample by lyophilization or rapid evaporation of a solution preparedfrom the crystalline sulfate. The amorphous solid sulfate material isthen dispersed in an aqueous solvent system, for example 25% water and75% acetonitrile and optionally agitated for a time period, greater thanabout 12 or greater than about 24 hours, at temperatures in the range ofabout 0 to about 65° C. Typically, the temperature is first raised toabout 65° C. and then lowered to between about 5 and about 20° C. Theresulting crystalline hydrate form is recovered conventionally.

Accordingly, in a method aspect, among other processes, the inventionprovides a process for preparing a crystalline sulfate salt of compound1, the process comprising: (a) contacting a protected precursor tocompound 1 in which the hydroxy groups are protected, with sulfuric acidto form a first reaction mixture; (b) isolating an intermediate gradesolid sulfate salt of compound 1 from the first reaction mixture; (c)dispersing the intermediate grade solid sulfate salt in a diluentcomprising methanol to form a second reaction mixture; and (d) isolatingthe crystalline sulfate salt from the second reaction mixture.

Further, in a composition aspect, the invention provides theN-cyclohexylmethyl-(2-oxoethyl)-carbamic acid benzyl ester bisulfiteadduct 3 useful for preparing compound 1. As described in Preparation 1,the bisulfite adduct 3 can be prepared by reductive amination ofcyclohexanecarboxaldehyde by 2,2-diethoxyethylamine using sodiumtriacetoxyborohydride, followed by addition of an amino-protectinggroup, deprotection of the aldehyde functionality, and conversion to thebisulfite adduct by reaction with sodium bisulfite. Alternatively, theinitial reductive amination can be performed by catalytic hydrogenation.Suitable hydrogenation catalysts, include, but are not limited to,palladium, platinum, and Raney nickel catalysts.

Pharmaceutical Compositions

The crystalline sulfate salt forms of the invention are typicallyadministered to a patient in the form of a pharmaceutical composition orformulation. Such pharmaceutical compositions may be administered to thepatient by any acceptable route of administration including, but notlimited to, oral, rectal, vaginal, nasal, inhaled, topical (includingtransdermal) and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a crystalline sulfate salt of compound 1 or asolvate thereof. Optionally, such pharmaceutical compositions maycontain other therapeutic and/or formulating agents if desired. Whendiscussing compositions, it is understood the term “salt of theinvention” includes the crystalline sulfate salt of compound 1 as wellas solvates, in particular, the hydrate, thereof.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of the active agent, present in theform of a salt of the invention. Typically, such pharmaceuticalcompositions will contain from about 0.1 to about 95% by weight of theactive agent; preferably, from about 5 to about 70% by weight; and morepreferably from about 10 to about 60% by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like, or unit packages suitablefor parenteral administration.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of a compound of the presentinvention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise the active agent and one ormore pharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylic acid estercopolymers, cellulose acetate trimellitate, carboxymethyl ethylcellulose, hydroxypropyl methyl cellulose acetate succinate, and thelike.

Pharmaceutical compositions of the invention may also be formulated toprovide slow or controlled release of the active agent using, by way ofexample, hydroxypropyl methyl cellulose in varying proportions; or otherpolymer matrices, liposomes and/or microspheres. In addition, thepharmaceutical compositions of the invention may optionally containopacifying agents and may be formulated so that they release the activeingredient only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active agent can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions, inaddition to the active ingredient, may contain suspending agents suchas, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The salts of this invention can also be administered parenterally (e.g.by intravenous, subcutaneous, intramuscular or intraperitonealinjection). For parenteral administration, the active agent is typicallyadmixed with a suitable vehicle for parenteral administration including,by way of example, sterile aqueous solutions, saline, low molecularweight alcohols such as propylene glycol, polyethylene glycol, vegetableoils, gelatin, fatty acid esters such as ethyl oleate, and the like.Parenteral formulations may also contain one or more anti-oxidants,solubilizers, stabilizers, preservatives, wetting agents, emulsifiers,or dispersing agents. These formulations may be rendered sterile by useof a sterile injectable medium, a sterilizing agent, filtration,irradiation, or heat.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The salts of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, theactive agent can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

If desired, the salts of this invention may be administered incombination with one or more other therapeutic agents. In thisembodiment, a salt of this invention is either physically mixed with theother therapeutic agent to form a composition containing both agents; oreach agent is present in separate and distinct compositions which areadministered to the patient simultaneously or sequentially.

For example, a salt of the invention can be combined with secondtherapeutic agent using conventional procedures and equipment to form acomposition comprising a compound 1 and a second therapeutic agent.Additionally, the therapeutic agents may be combined with apharmaceutically acceptable carrier to form a pharmaceutical compositioncomprising a salt of the invention, a second therapeutic agent and apharmaceutically acceptable carrier. In this embodiment, the componentsof the composition are typically mixed or blended to create a physicalmixture. The physical mixture is then administered in a therapeuticallyeffective amount using any of the routes described herein.Alternatively, the therapeutic agents may remain separate and distinctbefore administration to the patient. In this embodiment, the agents arenot physically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together as akit. The two therapeutic agents in the kit may be administered by thesame route of administration or by different routes of administration.

Any therapeutic agent compatible with the present active agent may beused as the second therapeutic agent. In particular, prokinetic agentsacting via mechanisms other than mu opioid receptor antagonism may beused in combination with the present compounds. For example, 5-HT₄receptor agonists, such as tegaserod, renzapride, mosapride,prucalopride, 1-isopropyl-1H-indazole-3-carboxylic acid{(1S,3R,5R)-8-[2-(4-acetylpiperazin-1-yl)ethyl]-8-azabicyclo[3.2.1]oct-3-yl}amide,1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide,or4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester may be used as the second therapeutic agent.

Additional useful prokinetic agents include, but are not limited to,5-HT₃ receptor agonists (e.g. pumosetrag), 5-HTIA receptor antagonists(e.g. AGI 001), alpha-2-delta ligands (e.g. PD-217014), chloride channelopeners (e.g. lubiprostone), dopamine antagonists (e.g. itopride,metaclopramide, domperidone), GABA-B agonists (e.g. baclofen, AGI 006),kappa opioid agonists (e.g. asimadoline), muscarinic M₁ and M₂antagonists (e.g. acotiamide), motilin agonists (e.g. mitemcinal),guanylate cyclase activators (e.g. MD-1100) and ghrelin agonists (e.g.Tzp 101, RC 1139).

In addition, the salts of the invention can be combined with opioidtherapeutic agents. Such opioid agents include, but are not limited to,morphine, pethidine, codeine, dihydrocodeine, oxycontin, oxycodone,hydrocodone, sufentanil, fentanyl, remifentanil, buprenorphine,methadone, and heroin.

Numerous additional examples of such therapeutic agents are known in theart and any such known therapeutic agents may be employed in combinationwith the compounds of this invention. Secondary agent(s), when included,are present in a therapeutically effective amount, i.e. in any amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. Suitable doses for the othertherapeutic agents administered in combination with a compound of theinvention are typically in the range of about 0.05 μg/day to about 100mg/day.

Accordingly, the pharmaceutical compositions of the invention optionallyinclude a second therapeutic agent as described above.

The following examples illustrate representative pharmaceuticalcompositions of the present invention:

FORMULATION EXAMPLE A Hard Gelatin Capsules for Oral Administration

A salt of the invention (50 g), spray-dried lactose (200 g) andmagnesium stearate (10 g) are thoroughly blended. The resultingcomposition is loaded into a hard gelatin capsule (260 mg of compositionper capsule).

FORMULATION EXAMPLE B Hard Gelatin Capsules for Oral Administration

A salt of the invention (20 mg), starch (89 mg), microcrystallinecellulose (89 mg), and magnesium stearate (2 mg) are thoroughly blendedand then passed through a No. 45 mesh U.S. sieve. The resultingcomposition is loaded into a hard gelatin capsule (200 mg of compositionper capsule).

FORMULATION EXAMPLE C Gelatin Capsules for Oral Administration

A salt of the invention (10 mg), polyoxyethylene sorbitan monooleate (50mg), and starch powder (250 mg) are thoroughly blended and then loadedinto a gelatin capsule (310 mg of composition per capsule).

FORMULATION EXAMPLE D Tablets for Oral Administration

A salt of the invention (5 mg), starch (50 mg), and microscrystallinecellulose (35 mg) are passed through a No. 45 mesh U.S. sieve and mixedthoroughly. A solution of polyvinylpyrrolidone (10 wt % in water, 4 mg)is mixed with the resulting powders, and this mixture is then passedthrough a No. 14 mesh U.S. sieve. The granules so produced are dried at50-60° C. and passed through a No. 18 mesh U.S. sieve. Sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg) and talc (1mg), which have previously been passed through a No. 60 mesh U.S. sieve,are then added to the granules. After mixing, the mixture is compressedon a tablet machine to afford a tablet weighing 100 mg.

FORMULATION EXAMPLE E Tablets for Oral Administration

A salt of the invention (25 mg), microcrystalline cellulose (400 mg),fumed silicon dioxide (10 mg), and stearic acid (5 mg) are thoroughlyblended and then compressed to form tablets (440 mg of composition pertablet).

FORMULATION EXAMPLE F Single-Scored Tablets for Oral Administration

A salt of the invention (15 mg), cornstarch (50 mg), croscarmellosesodium (25 mg), lactose (120 mg), and magnesium stearate (5 mg) arethoroughly blended and then compressed to form single-scored tablet (215mg of compositions per tablet).

FORMULATION EXAMPLE G Suspension for Oral Administration

The following ingredients are thoroughly mixed to form a suspension fororal administration containing 100 mg of active ingredient per 10 mL ofsuspension:

Ingredients Amount Salt of the invention 0.1 g Fumaric acid 0.5 g Sodiumchloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g Granulatedsugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k (Vanderbilt Co.)1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilled water q.s. to 100 mL

FORMULATION EXAMPLE H Dry Powder Composition

A micronized salt of the invention (1 mg) is blended with lactose (25mg) and then loaded into a gelatin inhalation cartridge. The contents ofthe cartridge are administered using a powder inhaler.

FORMULATION EXAMPLE J Injectable Formulation

A salt of the invention (0.1 g) is blended with 0.1 M sodium citratebuffer solution (15 mL). The pH of the resulting solution is adjusted topH 6 using 1 N aqueous hydrochloric acid or 1 N aqueous sodiumhydroxide. Sterile normal saline in citrate buffer is then added toprovide a total volume of 20 mL.

It will be understood that any form of salt of the invention, (i.e.crystalline salt, or solvate) that is suitable for the particular modeof administration, can be used in the pharmaceutical compositionsdiscussed above.

Utility

The present active agent,3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate, is an antagonist at the mu opioid receptor and therefore thesalts of the invention are expected to be useful for treating medicalconditions mediated by mu opioid receptors or associated with mu opioidreceptor activity, i.e. medical conditions which are ameliorated bytreatment with a mu opioid receptor antagonist. In particular, the saltsof the invention are expected to be useful for treating adverse effectsassociated with use of opioid analgesics, i.e. symptoms such asconstipation, decreased gastric emptying, abdominal pain, bloating,nausea, and gastroesophageal reflux, termed collectively opioid-inducedbowel dysfunction. The present salt forms are also expected to be usefulfor treating post-operative ileus, a disorder of reduced motility of thegastrointestinal tract that occurs after abdominal or other surgery. Inaddition, it has been suggested that mu opioid receptor antagonistcompounds, such as compound 1 may be used for reversing opioid-inducednausea and vomiting.

Since compound 1 has been shown to increase motility of thegastrointestinal (GI) tract in animal models, the salts of the inventionare expected to be useful for treating disorders of the GI tract causedby reduced motility in mammals, including humans. Such GI motilitydisorders include, by way of illustration, chronic constipation,constipation-predominant irritable bowel syndrome (C-IBS), diabetic andidiopathic gastroparesis, and functional dyspepsia.

In one aspect, therefore, the invention provides a method of increasingmotility of the gastrointestinal tract in a mammal, the methodcomprising administering to the mammal a therapeutically effectiveamount of a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a salt of the invention.

When used to treat disorders of reduced motility of the GI tract orother conditions mediated by mu opioid receptors, the salts of theinvention will typically be administered orally in a single daily doseor in multiple doses per day, although other forms of administration maybe used. For example, particularly when used to treat post-operativeileus, the compounds of the invention may be administered parenterally.The amount of active agent administered per dose or the total amountadministered per day will typically be determined by a physician, in thelight of the relevant circumstances, including the condition to betreated, the chosen route of administration, the actual compoundadministered and its relative activity, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thelike.

Suitable doses for treating disorders of reduced motility of the GItract or other disorders mediated by mu opioid receptors will range fromabout 0.0007 to about 20 mg/kg/day of active agent, including from about0.0007 to about 1.4 mg/kg/day. For an average 70 kg human, this wouldamount to from about 0.05 to about 100 mg per day of active agent.

In one aspect of the invention, the compounds of the invention are usedto treat opioid-induced bowel dysfunction. When used to treatopioid-induced bowel dysfunction, the compounds of the invention willtypically be administered orally in a single daily dose or in multipledoses per day. Preferably, the dose for treating opioid-induced boweldysfunction will range from about 0.05 to about 100 mg per day.

In another aspect of the invention, the compounds of the invention areused to treat post-operative ileus. When used to treat post-operativeileus, the compounds of the invention will typically be administeredorally or intravenously in a single daily dose or in multiple doses perday. Preferably, the dose for treating post-operative ileus will rangefrom about 0.05 to about 100 mg per day.

The invention also provides a method of treating a mammal having adisease or condition associated with mu opioid receptor activity, themethod comprising administering to the mammal a therapeuticallyeffective amount of a compound of the invention or of a pharmaceuticalcomposition comprising a compound of the invention.

The present active agent is optionally administered in combination withanother therapeutic agent or agents, in particular, in combination withprokinetic agents acting via non-mu opioid mechanisms. Accordingly, inanother aspect, the methods and compositions of the invention furthercomprise a therapeutically effective amount of another prokinetic agent.

As described above, salts of the invention are mu opioid receptorantagonists. The invention further provides, therefore, a method ofantagonizing a mu opioid receptor in a mammal, the method comprisingadministering a salt of the invention to the mammal.

Among other properties, the present active agent in freebase and sulfatesalt form has been found to exhibit potent binding to mu opioidreceptors and little or no agonism in mu receptor functional assays.Therefore, the salts of the invention are potent mu opioid receptorantagonists. Further, active agent has demonstrated predominantlyperipheral activity as compared with central nervous system activity inanimal models. Therefore, the salts of the invention can be expected toreverse opioid-induced reductions in GI motility without interferingwith the beneficial central effects of analgesia. These properties, aswell as the utility of the compounds of the invention, can bedemonstrated using various in vitro and in vivo assays well-known tothose skilled in the art. Representative assays are described in furtherdetail in the following examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

-   -   DIPEA=N,N-diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   EtOAc=ethyl acetate    -   EtOH=ethanol    -   MeTHF=2-methyltetrahydrofuran    -   MTBE=tert-butyl methyl ether    -   NaHMDS=sodium bis(trimethylsilyl)amide    -   PyBop=benzotriazol-1-yloxytripyrrolidino-phosphonium        hexafluorophosphate    -   psi=pounds per square inch    -   Rt=retention time    -   THF=tetrahydrofuran

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Reactionswere run under nitrogen atmosphere, unless noted otherwise. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Endo/exo ratios of products were determined by HPLCanalysis using the protocols described below. Reaction mixtures wereworked up as described specifically in each reaction; commonly they werepurified by extraction and other purification methods such astemperature-, and solvent-dependent crystallization, and precipitation.Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR measurement, samples were dissolved indeuterated solvent (CD₃OD, CDCl₃, or DMSO-d₆), and ¹H-NMR spectra wereacquired with a Varian Gemini 2000 instrument (300 MHz) under standardobservation conditions. Mass spectrometric identification of compoundswas performed by an electrospray ionization method (ESMS) with anApplied Biosystems (Foster City, Calif.) model API 150 EX instrument oran Agilent (Palo Alto, Calif.) model 1100 LC/MSD instrument.

General HPLC Conditions

Column: Zorbax SB-Aq, 5 μm. 4.6 × 250 mm Column temperature: 40° C. Flowrate: 1.0 mL/min Mobile Phases: A = Water/ACN (98:2) + 0.1% TFA B =Water/ACN (10:90) + 0.1% TFA, Injection volume: 10 μL Detectorwavelength: 214 nm

HPLC Method 1

Crude compounds were dissolved in Water/ACN (50:50) at about 1 mg/mL andanalyzed using the following gradient over 20 min (time (min)/% B):0/10, 2.5/20, 9/75, 15/90, 17/90, 18/10, 20/10.

HPLC Method 2

Compounds were dissolved in Water/ACN (90:10) at about 1 mg/mL andanalyzed using the following gradient over 30 min (time (min)/% B):0/10, 13/10, 23/65, 28/90, 29/90, 30/10.

HPLC Method 3

Compounds were dissolved in Water/ACN (90:10) at about 1 mg/mL andanalyzed using the following gradient over 55 min (time (min)/% B):0/10, 10/20, 46/75, 47/90, 50/10, 55/10.

Preparation 1: Synthesis of N-cyclohexylmethyl-(2-oxoethyl)-carbamicacid benzyl ester bisulfite adduct a. Preparation ofN-cyclohexylmethyl-(2,2-diethoxyethyl)amine

To a mixture of 2,2-diethoxyethylamine (209 mL, 1.43 mol) and MeTHF(1050 L) was added cyclohexanecarbaldehyde (107 mL, 0.89 mol). Thereaction mixture was stirred for 30 min at room temperature and cooledto 0° C. Sodium triacetoxyborohydride (378 g, 1.79 mol) was added over40 min and the reaction mixture was stirred for 2 h and cooled to 0° C.1 M NaOH (1 L) was added. The organic layer was washed with brine inwater (1:1, 2×1 L) and the volume was reduced to ˜20%. MeTHF (1 L) wasadded and the volume reduced to ˜20%. The solution of the crude titleintermediate was used directly in the next step.

b. Preparation of N-cyclohexylmethyl-(2,2-diethoxyethyl)carbamic acidbenzyl ester

To the product of the previous step (˜213 g, ˜0.9 mol) was added MeTHF(2 L) and DIPEA (233 mL, 1.34 mol). The reaction mixture was cooled to0° C. and benzylchloroformate (140 mL, 0.98 mol) was added dropwise. Thereaction mixture was stirred for 30 min at 0° C., for 2 h at 0° C. toroom temperature, and then for 1 h at room temperature. Water (1.6 L)was added and the reaction mixture was stirred for 10 min. The phaseswere separated and the organic layer was washed with sodium bicarbonate(1.6 L) and water (1.6 L). The layers were separated and the organiclayer was reduced to about 20%. MeTHF (1 L) was added and the volumereduced to ˜20%. The solution of the crude title intermediate was useddirectly in the next step.

c. Synthesis of N-cyclohexylmethyl-(2-oxoethyl)-carbamic acid benzylester bisulfite adduct

To the product of the previous step (˜302 g, ˜0.62 mol) and acetonitrile(2 L) was added 1 M HCl (2 L) and the reaction mixture was stirred at30° C. for 7 h. Ethyl acetate (2 L) was added and the reaction mixturewas stirred for 10 min. The phases were separated, the organic layer waswashed with 1 M HCl (1.5 L), the phases were again separated and theorganic layer was washed with 0.5 M HCl (1 L). Sodium bisulfite (71.4 g,0.69 mol) was added and the reaction mixture was stirred overnight, andthen filtered. The reactor and filter cake were washed with ethylacetate (1 L). The resulting solution was dried in air for 2 h and undervacuum overnight to provide the title compound as a white solid (199g, >99% area purity by HPLC). The filtrate was treated by the sameprocedure to provide a second lot of the title compound (30 g).

Preparation 2: Synthesis of3-endo-(8-azabicyclo[3.2.1]oct-3-yl)-benzamide a. Preparation of8-benzyl-3-exo-(3-methoxyphenyl)-8-azabicyclo[3.2.1]octan-3-ol

To a 3 L flask was added cerous chloride powder (194 g, 0.79 mol). Theflask was flushed with nitrogen and THF (800 mL) was added. The reactionmixture was stirred at 25° C. for 1 h. To the mixture was added ˜1M3-methoxyphenyl magnesium bromide in THF (800 mL, 0.87 mol) dropwise.The resulting slurry was stirred at 3° C. for 1.5 hours. A solution of8-benzyl-8-aza-bicyclo[3.2.1]octan-3-one (120.4 g, 0.56 mol) in THF (200mL) was then added dropwise, while maintaining the internal temperatureat −5° C. The resulting solution was stirred for 15 min. The reactionmixture was added to a flask containing 6 N HCl (800 mL) maintaining thetemperature at 10° C . After solvent was removed by rotary evaporation,the reaction mixture was stirred at room temperature overnight. Thesolids were isolated by filtration, washed with 6N HCL (70 mL) andacetonitrile (3×70 mL), and dried to provide the HCl salt of the titleintermediate as an off-white solid (161 g).

b. Preparation of8-benzyl-3-(3-methoxyphenyl)-8-azabicyclo[3.2.1]oct-2-ene

To a 3 L flask was added8-benzyl-3-exo-(3-methoxyphenyl)-8-azabicyclo[3.2.1]octan-3-olhydrochloride (383.9 g, 1.06 mol), 6 M HCl (800 mL), and MeTHF (200 mL).The resulting slurry was heated at 70° C. for 2.5 h under nitrogen. Thereaction mixture was transferred to a 12 L reactor and cooled to 10° C.The reaction flask was washed with MeTHF (1 L) that was added to the 12L reactor. NaOH (50 wt % in water, 200 mL) was added and additional NaOH(50 wt %, 150 mL) was added in portions until pH ˜13 was reached. Thephases were separated, the water layer was extracted with MeTHF (1 L),and combined MeTHF layers were washed with brine (1 L). Solvent wasreduced by rotary evaporation at 30 to 40° C. yielding the titleintermediate (360 g) as a thick oil. EtOH (1.5 L) was added and thevolume was reduced to ˜500 mL and then adjusted to 1.8 L.

c. Preparation of 3-endo-(3-methoxyphenyl)-8-azabicyclo[3.2.1]octane

To 8-benzyl-3-(3-methoxyphenyl)-8-azabicyclo[3.2.1]oct-2-ene (in EtOH95%, 400 mL, 0.20 mol), prepared in the previous step, was added 6 M HCl(45 mL) and then MeTHF (50 mL). The reaction mixture was purged withnitrogen, heated to 40° C. and palladium on carbon (10 weight %, 8 g)was added. The reactor was pressurized with hydrogen (3×20 psi) and thenhydrogenated at 20 psi at 40° C. for 18 h. The reaction mixture wasfiltered through Celite, concentrated, washed with MeTHF (2×100 mL),filtered through a coarse glass filter, washed with MeTHF (10 mL) anddried on the filter to provide the HCl salt of the title intermediate aswhite solid (31 g, single isomer, (exo isomer undetectable by HPLC)). Anadditional 5.2 g of product was recovered from the mother liquor.

d. Preparation of 3-endo-(8-azabicyclo[3.2.1]oct-3-yl)-phenol

To a 500 mL flask was added3-endo-(3-methoxyphenyl)-8-azabicyclo[3.2.1]octane hydrochloride (115 g,0.45 mol) and hydrobromic acid (48 weight % in water, 100 mL, 0.88 mol).The mixture was heated to 120° C. and held at that temperature for 24 hwith stirring. Additional hydrobromic acid solution (25 mL) was addedand the reaction mixture was heated with stirring for 6 h and thencooled to 70° C. Acetonitrile (200 mL) was added and the resultingslurry was cooled to 10° C. and then filtered, and the filter cake waswashed with acetonitrile (50 mL) to yield the HBr salt of the titleintermediate (99 g, >99% pure) as a white granular solid.

e. Preparation of2,2,2-trifluoro-1-[3-endo-(3-hydroxyphenyl)-8-azabicyclo[3.2.1]oct-8-yl]ethanone

To a solution of 3-endo-(8-azabicyclo[3.2.1]oct-3-yl)-phenolhydrobromide (54.4 g, 0.19 mol), toluene (210 mL), and triethylamine (40mL, 0.29 mol), was added trifluoroacetic anhydride (54 mL, 0.38 mol)over 20 min. The reaction mixture was stirred at 40° C. for 2 h. Ethylacetate (370 mL) and brine in water (1:1, 265 mL) were added. Thereaction mixture was stirred for 15 min, the phases were separated. Tothe organic layer was added saturated sodium bicarbonate (300 mL) andthe mixture was stirred vigorously overnight. The phases were separatedand the organic layer was washed with brine in water (1:1, 265 mL) driedover sodium sulfate and most of the solvent was removed by rotaryevaporation. Toluene (100 mL) was added and the solvent removed byrotary evaporation to provide the crude title intermediate.

f. Preparation of trifluoromethanesulfonic acid3-endo-[8-(2,2,2-trifluoro-acetyl)-8-azabicyclo[3.2.1]oct-3-yl]phenylester

To a 500 mL flask was added the ethyl acetate solution (220 mL) of theintermediate of the previous step (32.8 g, 0.11 mol) and triethylamine(23 mL. 0.17 mol). The solution was cooled to 5° C. and trifluoromethanesulfonyl chloride (14 mL, 0.13 mol) was added dropwise. The mixture wasallowed to warm to 25° C. and stirred at that temperature for 1 h.Saturated sodium bicarbonate (200 mL) was added, the layers wereseparated, brine (150 mL) was added to the organic layer, the layerswere again separated, and solvent was removed from the organic layer toprovide the crude title intermediate.

g. Preparation of3-endo-[8-(2,2,2-trifluoroacetyl)-8-azabicyclo[3.2.1]oct-3-yl]-benzonitrile

To a 100 mL flask was added trifluoromethanesulfonic acid3-endo-[8-(2,2,2-trifluoro-acetyl)-8-azabicyclo[3.2.1]oct-3-yl]phenylester (25.3 g, 58.7 mmol), tris(dibenzylideneacetone)dipalladium (0)(0.81 g, 0.9 mmol), 1,1′-bis(diphenylphosphino)ferrocene (1.01 g, 1.8mmol), and zinc cyanide (4.2 g, 35.8 mmol). Three times, the flask waspurged with nitrogen for 5 min and then placed under house vacuum for 5min. To the flask was added DMF (150 mL) and distilled water (2.5 mL).The solution was purged with nitrogen with stirring for 10 min, heatedto 120° C. and stirred at 120° C. under nitrogen for 4 h. When thereaction was completed 20 g of product from a previous lot, prepared bythe same procedure, was added and stirred for 20 min.

Most of the solvent was removed by distillation and the solution wascooled to 22° C. To the solution was added ethyl acetate (445 mL) andthe resulting solution was filtered through Celite. Sodium bicarbonate(450 mL) was added and the solution was stirred for 15 min. The layerswere separated and the organic layer was washed with diluted brine (2×95mL), and filtered through sodium sulfate. The volume was reduced toabout 50 mL by removal of ethyl acetate. Isopropyl alcohol (150 mL) wasadded and the solution was agitated at 22° C. for 1 h. Solids wereisolated by filtration and washed with isopropyl alcohol (2×25 mL) toprovide the title intermediate (33.5 g, 100% pure by HPLC) as anoff-white/light brown solid. A second crop of product (6.3 g, >98% pureby HPLC) was isolated from the filtrate.

h. Synthesis of 3-endo-(8-azabicyclo[3.2.1]oct-3-yl)-benzamide

A solution of3-endo-[8-(2,2,2-trifluoroacetyl)-8-azabicyclo[3.2.1]oct-3-yl]-benzonitrile(10 g, 32 mmol) in sulfuric acid (96%, 12 mL) was heated to 50° C. withstirring and held at that temperature with stirring for 2 h. Thereaction mixture was cooled to 22° C. and added slowly to a 500 mL flaskcontaining 5 N NaOH (90 mL) and methanol (100 mL) which was cooled to10° C. Salt precipitates were filtered and the filtrate was stirred at22° C. for 1 h. The reaction mixture was concentrated under reducedpressure. To the residue was added MeTHF (150 mL) and the reactionmixture was stirred at 22° C. for 5 min. The layers were separated andMeTHF (100 mL) was added to the aqueous layer. The layers were separatedand brine (150 mL) was added to the combined organic layers. The layerswere separated and the organic layer was dried over potassium carbonateand filtered, and the solvent was removed. A mixture of EtOH (25 mL) andconcentrated HCl (2.6 mL) was added to the residue with stirring andthen MTBE (25 mL) was added and the solution was stirred at 22° C.Precipitated solids were filtered and air dried to provide the HCl saltof the title compound (8 g, 97% purity by HPLC) as a white solid.

Preparation 3: Synthesis of3-endo-(8-azabicyclo[3.2.1]oct-3-yl)benzamide a. Preparation oftrifluoro-methanesulfonic acid 8-benzyl-8-azabicyclo[3.2.1]oct-2-en-3-ylester

To a 500 mL flask was added 8-benzyl-8-azabicyclo[3.2.1]octan-3-onehydrochloride (50.4 g, 200 mmol), EtOAc (160 mL), and 4 N NaOH (50 mL).The reaction mixture was heated to 30° C. and stirred at thattemperature for 1 h. The layers were separated and the aqueous layer wasdiscarded. The volume of the organic layer was reduced to ˜40 mL byrotary evaporation and THF (270 mL) was added.

The resulting solution was added to a 1 L flask and cooled to −20° C. ANaHMDS solution (1 M in THF, 230 mL, 230 mmol) was added to the flaskover 15 min. The reaction mixture was stirred at −20±5° C. for 1 h.N-phenyl-bis(trifluoromethanesulfonimide (82.2 g, 230 mmol) was added tothe reaction mixture in portions over 5 min and the mixture was stirredat −20° C. to −10° C. for 1 h. To the reaction mixture was added 1 NNaOH (200 mL) and the mixture was allowed to warm to 22° C. withstirring. Solvent was partially removed by rotary evaporation at 30° C.to a volume of 450 mL. To the remaining reaction mixture was added EtOAc(300 mL) and heptane (150 mL). The mixture was stirred at 22° C. for 5min. The layers were separated and the aqueous layer was discarded. Theorganic layer was washed with 1N NaOH (3×450 mL). The aqueous layerswere discarded. The organic layer was concentrated by rotary evaporationto provide the title intermediate (77 g, >96% purity by HPLC method 1).

¹H NMR (d₆-DMSO, 400 MHz): δ (ppm) 7.25-7.35 (m, 5H), 6.05 (d, J=5.2,1H), 3.64 (q, J=13.2, 2H), 3.40-3.44 (m, 2H), 2.77 (d, J=16.4, 1H),1.79-2.09 (m, 5H), 1.52-1.59 (m, 1H).

b. Preparation of 3-(8-benzyl-8-azabicyclo[3.2.1]oct-2-en-3-yl)benzamide

To the crude product of the previous step was added THF (420 mL) and thesolution was purged with nitrogen for 5 min. To a 2 L flask was added3-carbamoylphenyl boronic acid (98%, 33.0 g, 200 mmol), palladium (II)acetate (98%. 0.46 g, 2 mmol), 1,1′-bis(diphenylphosphino)ferrocene(97%, 1.1 g, 2 mmol). and potassium fluoride (34.9 g, 600 mmol) followedby the THF solution of trifluoro-methanesulfonic acid8-benzyl-8-aza-bicyclo[3.2.1]oct-2-en-3-yl ester. The resulting mixturewas purged with nitrogen for 5 min, heated to reflux (67° C.) undernitrogen and stirred for 2 h. The reaction mixture was cooled to 30° C.,then EtOAc (500 mL) and 1 N NaOH (500 mL) were added and the mixture wasstirred at 22° C. for 10 min. The layers were separated and the aqueouslayer was discarded. The organic layer was washed with a mixture ofbrine (250 mL) and water (250 mL) and stirred for 5 min. The layers wereseparated and the aqueous layer was discarded. The organic layer wasbriefly dried over Na₂SO₄, filtered, and solvent was partially removed.Product precipitated as light yellow solids during solvent removal. Theresulting slurry (about 200 mL) was filtered and the solids were washedwith cold EtOAc (0° C., 100 mL) and dried under high vacuum at 25° C. toprovide the title intermediate (42.5 g) as a light yellow solid.

The mother liquor and the above washes were combined and concentratedand the resulting slurry (about 100 mL) was stirred at 5° C. for 30 minand filtered. The filtered solids were washed with cold EtOAc (0° C., 30mL) and dried under high vacuum to provide a second crop of the titleintermediate (7 g, combined yield 78%, >98.5% pure by HPLC method 1).

(m/z): [M+H]⁺ calcd for C₂₁H₂₂N₂O, 319.18; found 319.4. ¹H NMR (CDCl₃,400 MHz): δ (ppm) 7.9 (s, 1H), 7.63 (d, J=6.4, 1H), 7.57 (d, J=6.4, 1H),7.21-7.42 (m, 6H), 6.38 (d, J=4.4, 1H), 6.13 (s, br, 1H), 5.83 (s, br,1H), 3.68-3.76 (m, 2H), 3.46-3.51 (m, 2H), 2.92 (d, J=17.2, 1H),2.18-2.26 (m, 1H), 2.04-2.12 (m, 2H), 1.86-1.92 (m, 1H), 1.58-1.65 (m,1H).

c. Synthesis of 3-endo-(8-azabicyclo[3.2.1]oct-3-yl)benzamide

To a 1 L hydrogenation vessel was added3-(8-benzyl-8-azabicyclo[3.2.1]oct-2-en-3-yl)benzamide (40 g, 125 mmol),EtOH (800 mL), 6 M HCl (42 mL) and water (80 mL) and the mixture wasstirred at 22° C. until complete dissolution was observed. The reactionmixture was purged with nitrogen for 5 min while being heated to 30° C.over 5 min. To the mixture was added 10 wt % Pd/C (50% in water, 4 g).The mixture was purged at atmospheric pressure with hydrogen for 5-10min while being heated. The mixture was stirred at 50° C. under a flowof hydrogen at <5 psi (<0.34 atmospheres) for 5 h, resulting in >99%conversion of the reactants, according to HPLC analysis. The solutionwas cooled to 30° C. and filtered through Celite to provide a solutionof the crude HCl salt of the title compound with an endo:exo ratio of˜93:7 by HPLC method 2 endo Rt=10.97, exo Rt=12.67. (m/z): [M+H]⁺ calcdfor C₁₄H₁₈N₂O, 231.15; found 231.2.

Water was removed from the crude product by azeotropic distillation at30° C. in EtOH (˜80 mL) to provide a slurry that was heated to 60° C.until complete dissolution. The solution was cooled to 35° C. and seedcrystals of the product (0.05 g) were added. (The seed crystals wereprepared according to the process described in Preparation 2.) Theresulting slurry was stirred at 22° C. for 30 min, MTBE (120 mL) wasadded slowly, and the slurry was stirred at 22° C. for 4 h and then at0° C. for 1 h. The resulting solids were filtered, washed with cold EtOHand dried under high vacuum to provide the HCl salt of the titlecompound (24.5 g) as a white powder (75% yield, >98.5% purity <0.4% exoisomer by HPLC method 3, endo Rt=8.67, exo Rt=9.43).

(m/z): [M+H]⁺ calcd for C₁₄H₁₈N₂O 231.15; found 231.2. ¹H NMR (d₆-DMSO,400 MHz): δ (ppm) 9.13 (s, br, 1H), 9.03 (s, br, 1H), 8.05 (s, 1H), 7.93(s, 1H), 7.73 (d, J=7.6, 1H), 7.58 (d, J=7.6, 1H), 7.40 (t, J=7.6, 2H),3.97 (s, 2H), 3.17-3.23 (m, 1H), 2.39-2.46 (m, 2H), 2.19-2.24 (m, 2H),1.86-1.89 (m, 2H), 1.59-1.63 (m, 2H).

Example 1A Synthesis of crystalline3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate a. Preparation of N-cyclohexylmethyl-(2-oxoethyl)-carbamic acidbenzyl ester

To a 100 mL flask was added N-cyclohexylmethyl-(2-oxoethyl)-carbamicacid benzyl ester bisulfite adduct (3.94 g, 1 mmol) and MeTHF (35 mL),followed by water (25 mL). The resulting slurry was stirred at roomtemperature for 5 min and 1 M NaOH (8 mL) was added. The reactionmixture was stirred at room temperature for 45 min. The layers wereseparated and the volume of the organic layer was reduced to ˜8 mL toprovide the crude title intermediate.

b. Preparation of2-[3-endo-(3-carbamoylphenyl)-8-azabicyclo[3.2.1]oct-8-yl]-ethyl}cyclohexylmethyl-carbamicacid benzyl ester

To the product of the previous step was added DMF (15 mL) followed by3-endo-(8-azabicyclo[3.2.1]oct-3-yl)-benzamide hydrochloride (2.67 g, 1mmol) and then DMF (10 mL). The mixture was stirred at room temperaturefor 30 min, cooled to 10° C. and then sodium triacetoxyborohydride (4.25g, 2 mmol) was added. The reaction mixture was stirred at roomtemperature for 90 min and then cooled to 10° C. Isopropyl acetate (100mL) was added, followed by 1 M NaOH (50 mL). The mixture was stirred for15 min, and the phases were separated. The organic layer was washed withbrine in water (1:1, 2×50 mL) and the volume of the organic layer wasreduced to ˜10 mL to provide the crude title intermediate.

c. Preparation of3-endo-{8-[2-(cyclohexylmethylamino)ethyl]-8-aza-bicyclo[3.2.1]oct-3-yl}benzamide

To the product of the previous step was added EtOH (30 mL) andconcentrated HCl (1.5 mL). The solution was purged with nitrogen, 10%palladium on carbon (470 mg) was added and the mixture was purged withnitrogen for 5 min and then hydrogenated at 30 psi overnight. Afterpurging with nitrogen for 2 min, the solution was filtered throughCelite and solvent was removed to ˜10 mL. Isopropyl acetate (40 mL) and1 M NaOH (20 mL) were added. The layers were separated and the organiclayer was washed with brine (20 mL), phases were separated and organicsolvent removed to 5-10 mL. Isopropyl acetate (20 mL) was added and thevolume reduced to ˜8 mL to which isopropyl acetate (20 mL) was added.The resulting slurry was stirred at room temperature for 2 h. Theproduct was isolated by filtration, the reaction flask and filter cakewere washed with isopropyl acetate (10 mL) to yield the titleintermediate (2.4 g, 98% pure) as an off-white solid.

d. Preparation of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate (hydrate form)

To a 500 mL flask was added3-endo-{8-[2-(cyclohexylmethylamino)ethyl]-8-aza-bicyclo[3.2.1]oct-3-yl}benzamide(31 g, 83.9 mmol) and DMF (150 mL). The mixture was stirred for 10 minand then benzotriazol-1-yloxytris(pyrrolidino)-phosphoniumhexafluoro-phosphate (56.8 g, 109 mmol) and lithium(4S)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (15.6 g, 92.3 mmol) wereadded and the mixture was stirred at room temperature for 2 h. Ethylacetate (600 mL) and 0.5 M NaOH (300 mL) were added and the phases wereseparated. The organic layer contained crude(S)-2,2-dimethyl-[1,3]dioxolane-4-carboxylic acid{2-[3-(3-carbamoyl-phenyl)-8-azabicyclo[3.2.1]oct-8-yl]ethyl}cyclohexylmethyl-amide(˜84 mmol) which was not isolated.

The organic layer was washed with brine in water (1:1, 2×300 mL) and thephases were separated. To the organic layer was added 2 M H₂SO₄ (42 mL)and the reaction mixture was stirred at room temperature overnight.Acetonitrile (300 mL) was added and the resulting slurry was stirred for2 h. The product was isolated by filtration, the filter cake was washedwith acetonitrile (200 mL), dried in air for 2 h and then under vacuumat room temperature for 20 h to provide the title compound (40 g, 97%pure by HPLC) as a white powder.

e. Synthesis of crystalline3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate

To a 100 mL flask was added3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate hydrate form (2 g) and MeOH (40 mL). The resulting slurry washeated to 65° C. under nitrogen for 20 min resulting in completedissolution. The solution was cooled to room temperature with stirring.About 20 mL of solvent was removed under slightly reduced pressure andthe resulting slurry stirred at room temperature overnight. The productwas isolated by filtration, and the flask and filter cake were washedwith acetonitrile (2×5 mL). The filter cake was dried in air for 2 h andthen under vacuum at room temperature overnight to provide the titlecompound (1.71 g, >99% pure by HPLC, ˜85% yield) as a white powder.

A sample prepared according to the above procedure was characterized by¹H NMR (400 MHz, DMSO d₆): δ (ppm) 9.08 & 8.94 (two sets of brs, 1H),7.99-8.04 (m, 2H), 7.74-7.76 (m, 1H), 7.68-7.70 (m, 1H), 7.41-7.45 (m,2H), 4.81, 5.00 and 5.30 (three sets of brs, 2H), 4.34 (deformed m, 1H),4.00 & 4.05 (deformed m, 2H), 3.01-3.25 and 3.47-3.55 and 3.75-3.82(three sets of m, 10H), 2.50-2.55 (m, 2H), 1.99 (deformed m, 2H),1.56-1.70 (m, 8H), 1.15-1.19 (m, 3H), 0.89-0.99 (m, 2H).

Example 1B Synthesis of crystalline3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate a. Preparation of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate

A mixture of3-endo-{8-[2-(cyclohexylmethylamino)ethyl]-8-aza-bicyclo[3.2.1]oct-3-yl}benzamide(100 g, 270.6 mmol) and DMF (480 mL) was stirred for 10 min and thencooled at 0° C. Benzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate (183 g, 352 mmol) and lithium(4S)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (49.3 g, 324 mmol) wereadded in one portion at 0° C. The reaction mixture was stirred at roomtemperature for 6 h. Isopropyl acetate (2.0 L) and 1 M NaOH (1.0 L) wereadded, the reaction mixture was stirred for 15 min, and the phases wereseparated. The organic layer was washed with brine in water (1:1, 2×1.0L) and the phases were separated. The organic layer was reduced to aquarter of the volume (˜500 mL), acetonitrile (500 mL) was added and thereaction mixture was stirred until homogenous to provide a solution ofintermediate (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid2-[3-endo-(3-carbamoyl-phenyl)-8-azabicyclo[3.2.1]oct-8-yl]-ethyl-cyclohexylmethyl-amidein isopropyl acetate and acetonitrile.

An aliquot of the above solution of intermediate (3.03 g, 6.09 mmol) inisopropyl acetate/acetonitrile (22.5 mL) was combined with 2.0 Msulfuric acid in water (3.68 mL) and held at 25° C. for 20 h and thenheld at 10° C. with stirring for 5 h. The reaction solution wasfiltered, and the filter cake was washed with acetonitrile (25 mL) anddried to yield the intermediate grade sulfate salt of the title compound(2.91 g, 99.4% purity by HPLC) as a white solid, predominantly incrystalline hydrate form.

b. Synthesis of crystalline3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydrox-propionyl)aminolethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate

A mixture of3-endo-(8-2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)-amino]-ethyl-8-aza-bicyclo[3.2.1]oct-3-yl)-benzamidesulfate intermediate grade (154.0 g, 277.1 mmol) prepared as in theprevious step and methanol/10% water (616 mL) was heated at 65° C. over45 min with stirring. The reaction mixture was cooled to 55° C., seedcrystals of the title product (120 mg) were added, the reaction mixturewas stirred at 55° C. for 1 h, and the temperature was reduced to 20° C.at the rate of 10° C./h and then held for 8 h. The reaction mixture wascooled to 5° C., held for 30 min, and filtered. The filter cake waswashed with methanol (2×25 mL) and dried overnight under high vacuum toyield the title compound (126.3 g, 99.9% purity)

Example 2 Recrystallization of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate (hydrate form)

Compound 1 sulfate (hydrate form) (920 mg) was suspended in acetonitrile(5 mL) and heated to 65° C. Water (2.4 mL) was then added dropwise untilcomplete dissolution was achieved. The resulting solution was cooled toambient temperature over 20 min. Nucleation was observed around 35° C.The solids were isolated by vacuum filtration, washed with acetonitrile(5 mL) and dried to provide the title compound.

Example 3 Crystallization of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate

Compound 1 sulfate (hydrate form) (50 mg) was dispersed in a water (10%)and methanol (90%) solvent mixture (0.83 mL) and heated to 60° C. withstirring. The resulting solution was allowed to cool to ambienttemperature over 2 h. The resulting solids were isolated by vacuumfiltration to provide the title compound (8 mg).

Example 4 Crystallization of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate

Compound 1 sulfate (hydrate form) (42 mg) was dispersed in a water (25%)and methanol (75%) solvent mixture (0.42 mL) and heated to 60° C. withstirring. The resulting solution was allowed to cool to ambienttemperature. The volume was reduced by 50% by rotary evaporation and thesolution was left at ambient temperature overnight. The resulting solidswere isolated by vacuum filtration to provide the title compound (8 mg).

Example 5 Crystallization of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate

Compound 1 (11 mg) was dissolved in a toluene (22%) and acetonitrile(78%) solvent mixture (0.2 mL). Acetonitrile (0.15 mL) was added,followed by 0.04 M sulfuric acid in acetonitrile (0.59 mL). A solidprecipitate formed on addition of acid. The reaction mixture was left atambient temperature for 12 h. The resulting solids were isolated byfiltration to provide the title compound.

Example 6 Crystallization of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate

Compound 1 (38 mg) was dissolved in dichloromethane (0.5 mL). To thesolution was added 0.04 M sulfuric acid in acetonitrile (1.91 mL). Asolid precipitate formed on addition of acid. The reaction mixture wasleft at ambient temperature for 12 h. The resulting solids were isolatedby filtration to provide the title compound.

Example 7 Crystallization of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate

Compound 1 (22 mg) was dissolved in a toluene (23%) and acetonitrile(77%) solvent mixture (0.41 mL). To the solution was added 0.04 Msulfuric acid in acetonitrile (1.20 mL). A solid precipitate formed onaddition of acid. Water (0.16 mL) was added to the reaction mixturedissolving the precipitate. Nucleation was observed after 2 h. Theresulting solids were isolated by vacuum filtration to provide the titlecompound.

Example 8 Crystallization of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidesulfate (hydrate form)

Crystalline compound 1 sulfate (7.1 g) was dissolved in a solventmixture of water (42 mL) and acetonitrile (25 mL). The solution waslyophilized to produce an amorphous sulfate salt. The amorphous salt(6.6 g) was dispersed in a acetonitrile (75%) and water (25%) solventmixture (34.6 mL) and heated to 65° C. for 10 min with stirring andallowed to cool with stirring until ambient temperature was reached.After 12 h, the resulting solids were isolated by vacuum filtration toprovide the title compound (5.4 g).

Examples 9-17 Properties of Salt Forms of the Invention

Samples of the crystalline sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide(compound 1) prepared as in Example 1A and of the crystalline hydrate ofthe sulfate salt of compound 1, prepared as in Example 2 were analyzedby x-ray powder diffraction (XRPD), differential scanning calorimetry(DSC), thermogravimetric analysis (TGA) infrared spectroscopy (IR) andion chromatography.

Example 10 X-Ray Powder Diffraction

X-ray powder diffraction patterns of FIGS. 1 and 4 were obtained with aRigaku diffractometer using Cu Ka (30.0 kV, 15.0 mA) radiation. Theanalysis was performed with the goniometer running in continuous-scanmode of 3° per min with a step size of 0.03° over a range of 2 to 40°.Samples were prepared on quartz specimen holders as a thin layer ofpowdered material. The instrument was calibrated with a siliconstandard.

Example 11 Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module. Data were collected and analyzed usingTA Instruments Thermal Advantage for Q Series™ software. A sample ofabout 1-10 mg was accurately weighed into an aluminum pan with lid. Thesample was evaluated using a linear heating ramp of 10° C./min from 5°C. to, typically, 265° C. The DSC cell was purged with dry nitrogenduring use. Representative DSC traces for samples of the crystallinesulfate salt and of the crystalline hydrate of a sulfate salt ofcompound 1 are shown in FIGS. 2 and 5, respectively.

Thermogravimetric analysis (TGA) was performed using a TA InstrumentsModel Q-500 module. Data were collected and analyzed using TAInstruments Thermal Advantage for Q Series™ software. A sample weighingabout 1-5 mg was placed in an aluminum pan on a platinum cradle andscanned from ambient temperature to 300° C. with a linear heating rateof 10° C./min. The balance and furnace chambers were purged withnitrogen during use. Representative TGA traces for samples of acrystalline sulfate salt and of a crystalline hydrate of a sulfate saltof compound 1 are also shown in FIGS. 2 and 5, respectively.

Example 12 Dynamic Moisture Sorption Assessment

Dynamic moisture sorption (DMS) assessment was performed at 25° C. usinga VTI atmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, Fla.33016). A sample size of approximately 5-10 mg was used and the humiditywas set at the ambient value at the start of the analysis. A typical DMSanalysis consisted of three scans: ambient to 2% relative humidity (RH),2% RH to 90% RH, 90% RH to 5% RH at a scan rate of 5% RH/step. The masswas measured every two minutes and the RH was changed to the next value(±5% RH) when the mass of the sample was stable to within 0.02% for 5consecutive points. Representative DMS traces for samples of acrystalline sulfate salt and of a crystalline hydrate of a sulfate saltof compound 1 are shown in FIGS. 3 and 6, respectively.

Example 13 Infrared Analysis

The infrared (IR) absorption spectrum was determined over the frequencyrange 4000 to 400 cm⁻¹ using an Avatar 360 FT-IR spectrometer equippedwith a diffuse reflectance infrared fourier transform spectroscopy(DRIFTS) module. A representative IR absorption spectrum for a sample ofa crystalline sulfate salt of the invention had significant absorptionbands at 430±1, 590±1, 639±1, 705±1, 867±1, 1036±1, 1053±1, 1105±1,1171±1, 1231±1, 1277±1, 1375±1, 1391±1, 1452±1, 1476±1, 1553±1, 1596±1,1639±1, 1664±1, 2852±1, 2907±1, 2928±1, 2967±1, 3168±1, and 3357±1 cm⁻¹.

Example 14 X-ray Diffraction Crystal Structure Analysis

A chunk crystal of the sulfate salt of compound 1 having dimensions of0.43×0.05×0.031 mm was mounted on a glass fiber. X-ray diffractioncrystal structure data was obtained using a Bruker SMART 6 K CCD x-rayarea detector with window diameter of 13.5 cm, controlled by SMARTversion 5.630 software (Bruker, 2003) using Cu Ka radiation. The sampleto detector distance was 5.039 cm. Data was collected at a temperatureof −153±1° C. and was analyzed using SHELXS version 6.14 (Bruker, 2003)software. The following lattice parameters were derived: unit cell isorthorhombic with dimensions a=6.8239 Å, b=16.2275 Å, c=24.2021 Å,α=β=γ=90°; cell volume (V) of 2680.0 Å³; calculated density is 1.38g/cm³; space group is P2₁2₁2₁(#19) Powder x-ray diffraction peakspredicted from the derived atomic positions according to Mercury 1.4software were judged by visual inspection to be in excellent agreementwith the experimental results of FIG. 1.

Example 15 Solid State Stability Assessment

Samples of the sulfate salt of the invention were stored in multipleopen glass vials at 20° C. and 60% relative humidity (RH) and at 40° C.and 75% RH. At specific intervals, the contents of a representative vialwas removed and analyzed by DSC, TGA, PXRD, and by HPLC for chemicalpurity. After 4 weeks of storage, there was no detectable change in theDSC or TGA thermograms nor in the XRPD pattern of samples stored ateither condition. The chemical purity of the stored samples by HPLC wasunchanged at 99.7%.

Example 16 Determination of Counterion Content

A sample of the sulfate salt of the invention was analyzed by sulfateion chromatography using a Dionex ICS-2000 ion chromatography systemequipped with an anion self-regenerating suppressor, conductivitydetector, IonPac AS11-HC analytical anion-exchange column, and IonPacAG11-HC guard column. The sulfate content of the sample was determinedto be 17.1% which may be compared with a theoretical sulfate content of17.6% for one molar equivalent of sulphate ion per mole of parentcompound.

Example 17 Determination of Water Content of Hydrate

A sample of the hydrate of the invention was analyzed by TGA coupledwith IR analysis of the material vaporized during initial weight loss.The TGA trace shows a weight loss of 3.2% below 100° C. which may becompared with a theoretical weight loss of 3.1% for a monohydrate ofcompound 1 sulfate. The IR spectrum of the vaporized material wasconsistent with the reference IR spectrum of water.

Assay 1: Radioligand Binding Assay on Human Mu, Human Delta and GuineaPig Kappa Opioid Receptors

a. Membrane Preparation

CHO-K1 (Chinese Hamster Ovary) cells stably transfected with human muopioid or with guinea pig kappa receptor cDNA were grown in mediumconsisting of Ham's-F12 media supplemented with 10% FBS, 100 units/mlpenicillin—100 μg/mL streptomycin and 800 μg/mL Geneticin in a 5% CO₂,humidified incubator @37° C. Receptor expression levels (B_(max)˜2.0 and˜0.414 pmol/mg protein, respectively) were determined using[³H]-Diprenorphine (specific activity 50-55 Ci/mmol) in a membraneradioligand binding assay.

Cells were grown to 80-95% confluency (<25 subculture passages). Forcell line passaging, the cell monolayer was incubated for 5 minutes atroom temperature and harvested by mechanical agitation in 10 mL of PBSsupplemented with 5 mM EDTA. Following resuspension, cells weretransferred to 40 mL fresh growth media for centrifugation for 5 minutesat 1000 rpm and resuspended in fresh growth medium at the appropriatesplit ratio.

For membrane preparation, cells were harvested by gentle mechanicalagitation with 5 mM EDTA in PBS followed by centrifugation (2500 g for 5minutes). The pellets were resuspended in Assay Buffer (50 mM4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acidN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES)), pH7.4, and homogenized with a polytron disrupter on ice. The resultanthomogenates were centrifuged (1200 g for 5 minutes), the pelletsdiscarded and the supernatant centrifuged (40,000 g for 20 minutes). Thepellets were washed once by resuspension in Assay Buffer, followed by anadditional centrifugation (40,000 g for 20 minutes). The final pelletswere resuspended in Assay Buffer (equivalent 1 T-225 flask/1 mL assaybuffer). Protein concentration was determined using a Bio-Rad BradfordProtein Assay kit and membranes were stored in frozen aliquots at −80°C., until required.

Human delta opioid receptor (hDOP) membranes were purchased from PerkinElmer. The reported K_(d) and B_(max) for these membranes determined bysaturation analyses in a [³H]-Natrindole radioligand binding assays were0.14 nM (pK_(d)=9.85) and 2.2 pmol/mg protein, respectively. Proteinconcentration was determined using a Bio-Rad Bradford Protein Assay kit.Membranes were stored in frozen aliquots at −80° C., until required.

b. Radioligand Binding Assays

Radioligand binding assays were performed in an Axygen 1.1 mL deep well96-well polypropylene assay plate in a total assay volume of 200 μLcontaining the appropriate amount of membrane protein (˜3, ˜2 and ˜20 μgfor mu, delta and kappa, respectively) in Assay Buffer, supplementedwith 0.025% bovine serum albumin (BSA). Saturation binding studies fordetermination of K_(d) values of the radioligand were performed using[³H]-Diprenorphine at 8-12 different concentrations ranging from 0.001nM-5 nM. Displacement assays for determination of pKi values ofcompounds were performed with [³H]-Diprenorphine at 0.5, 1.2, and 0.7 nMfor mu, delta, and kappa, respectively, and eleven concentrations ofcompound ranging from 10 μM-100 μM.

Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The curveminimum was fixed to the value for nonspecific binding, as determined inthe presence of 10 μM naloxone. K_(i) values for test compounds werecalculated, in Prism, from the best fit IC₅₀ values, and the K_(d) valueof the radioligand, using the Cheng-Prusoff equation(K_(i)=IC₅₀/(1+([L]/K_(d))) where [L]=the concentration of[³H]-Diprenorphine. Results are expressed as the negative decadiclogarithm of the K_(i) values, pK_(i).

Test compounds having a higher pK_(i) value in these assays have ahigher binding affinity for the mu, delta, or kappa opioid receptor. Thesulfate salt of compound 1 exhibited a pK_(i) value of 9.9 at the humanmu opioid receptor.

Assay 2: Agonist Mediated Activation of the mu-Opioid Receptor inMembranes Prepared from CHO-K1 Cells Expressing the Human mu-OpioidReceptor

In this assay, the potency and intrinsic activity values of testcompounds were determined by measuring the amount of bound GTP-Eupresent following receptor activation in membranes prepared from CHO-K1cells expressing the human mu opioid receptor.

a. Mu Opioid Receptor Membrane Preparation:

Human mu opioid receptor (hMOP) membranes were either prepared asdescribed above or were purchased from Perkin Elmer. The reported pK_(d)and B_(max) for the purchased membranes determined by saturationanalyses in a [³H]-Diprenorphine radioligand binding assays was 10.06and 2.4 pmol/mg protein, respectively. Protein concentration wasdetermined using a Bio-Rad Bradford Protein Assay kit. Membranes werestored in frozen aliquots at −80° C., until required. Lyophilized GTP-Euand GDP were diluted to 10 μM and 2 mM, respectively, in doubledistilled H₂O then mixed and permitted to sit at room temperature for 30minutes prior to transfer to individual aliquots samples for storage at−20° C.

b. Human mu GTP-Eu Nucleotide Exchange Assay

GTP-Eu nucleotide exchange assays were performed using the DELPHIAGTP-binding kit (Perkin/Elmer) in AcroWell 96 well filter platesaccording to the manufacturer's specifications. Membranes were preparedas described above, and prior to the start of the assay, aliquots werediluted to a concentration of 200 μg/mL in Assay Buffer (50 mM HEPES, pH7.4 at 25° C.), then homogenized for 10 seconds using a Polytronhomogenizer. Test compounds were received as 10 mM stock solutions inDMSO, diluted to 400 μM into Assay Buffer containing 0.1% BSA, andserial (1:5) dilutions then made to generate ten concentrations ofcompound ranging from 40 pM⁻ 80 μM-GDP and GTP-Eu were diluted to 4 μMand 40 nM, respectively, in Assay Buffer. The assay was performed in atotal volume of 100 μL containing 5 μg of membrane protein, testcompound ranging from 10 pM-20 μM), 1 μM GDP, and 10 nM GTP-Eu dilutedin 10 mM MgCl₂, 50 mM NaCl, and 0.0125% BSA, (final assayconcentrations). A DAMGO (Tyr-D-Ala-Gly-(methyl)Phe-Gly-ol)concentration-response curve (ranging from 12.8 pM-1 μM) was included onevery plate.

Assay plates were prepared immediately prior to assay following theaddition of 25 μL of Assay Buffer, 25 μL of test compound, and 25 μL GDPand GTP-Eu. The assay was initiated by the addition of 25 μL membraneprotein and allowed to incubate for 30 minutes. The assay plates werethen filtered with a Waters vacuum manifold connected to the housevacuum regulated to 10-12 in. Hg and washed with room temperature GTPWash Solution (2×300 mL). The bottoms of the plates were blotted toremove excess liquid. The plates were then immediately read to determinethe amount of bound GTP-Eu by measuring Time Resolved Fluorescence (TRF)on a Packard Fusion Plate ReaderVehicle: DMSO not to exceed 1% finalassay concentration.

The amount of bound GTP-Eu is proportional to the degree of activationof the mu opioid receptors by the test compound. The intrinsic activity(IA), expressed as a percentage, was determined as the ratio of theamount of bound GTP-Eu observed for activation by the test compound tothe amount observed for activation by DAMGO which is presumed to be afull agonist (IA=100). The sulfate salt of compound 1 demonstrated anintrinsic activity of −5 in this assay. Thus, the present sulfate salthas been shown to be an antagonist.

Assay 3: Rat Model of In Vivo Efficacy

In this assay the efficacy of test compounds was evaluated in a model ofgastrointestinal transit, which evaluates peripheral activity. Thisstudy was approved by the Institutional Animal Care and Use Committee atTheravance, Inc. and conformed to the Guide for the Care and Use ofLaboratory Animals published by the National Academy of Sciences©(1996).

a. Rat Gastric Emptying Assay

Test compounds were evaluated in the rat gastric emptying assay todetermine their ability to reverse loperamide-induced delayed gastricemptying. Rats were fasted up overnight prior to administration of testcompounds or vehicle by intravenous, subcutaneous, intramuscular or oralroutes of administration at doses ranging from 0.001 to about 30milligrams/kilogram (mg/kg). The administration of test compound wasfollowed by subcutaneous administration of loperamide at a dose of 1mg/kg or vehicle. Five minutes post loperamide or vehicleadministration, a non-nutritive, non-absorbable charcoal meal wasadministered via oral gavage and animals were allowed free access towater for the sixty minute duration of the experiment. Animals were theneuthanized via carbon dioxide asphyxiation followed by thoracotomy andthe stomach was carefully excised. The stomach was ligated at the loweresophageal sphincter and the pyloric sphincter to prevent additionalemptying during tissue removal. Gastric weight was then determined afterremoval of the ligatures.

b. Data Analysis and Results

Data was analyzed using the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.). Percent reversal curves wereconstructed by non-linear regression analysis using the sigmoidal doseresponse (variable slope) model and best-fit ID₅₀ values werecalculated. Curve minima and maxima were fixed to loperamide controlvalues (indicating 0% reversal) and vehicle controls (indicating 100%reversal), respectively. Results are expressed as ID₅₀, the doserequired for 50% reversal of the effects of loperamide, in milligramsper kilogram. The sulfate salt of compound 1, administered orally,exhibited an ID₅₀ value of 0.26 mg/kg in the gastric emptying model.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1. A crystalline salt form which is the sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamideor a solvate thereof.
 2. The crystalline salt form of claim 1 whereinthe crystalline salt form is a crystalline sulfate salt.
 3. Thecrystalline salt form of claim 2, wherein the crystalline salt form ischaracterized by an x-ray powder diffraction pattern having two or morediffraction peaks at 2θ values selected from 6.58±0.20, 7.52±0.20,9.35±0.20, 14.69±0.20, 16.01±0.20, 17.45±0.20, 17.99±0.20, 18.62±0.20,19.76±0.20, 21.11±0.20, 22.07±0.20, 23.18±0.20, 23.74±0.20, 24.56±0.20,25.63±0.20, 26.45±0.20, 27.86±0.20, 28.31±0.20, 29.54±0.20, 30.59±0.20,31.58±0.20, 33.89±0.20, and 36.02±0.20.
 4. The crystalline salt form ofclaim 3 wherein the x-ray powder diffraction pattern comprises two ormore diffraction peaks at 2θ values selected from 14.69±0.20,16.01±0.20, 21.11±0.20, 22.07±0.20, and 23.18±0.20.
 5. The crystallinesalt form of claim 2, wherein the crystalline salt form is characterizedby an x-ray powder diffraction pattern in which the peak positions aresubstantially in accordance with the peak positions of the pattern shownin FIG.
 1. 6. The crystalline salt form of claim 2, wherein thecrystalline salt form is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow at a temperature between about190° C. and about 205° C.
 7. The crystalline salt form of claim 2,wherein the crystalline salt form is characterized by a differentialscanning calorimetry trace substantially in accordance with that shownin FIG.
 2. 8. The crystalline salt form of claim 1 wherein thecrystalline salt form is a hydrate of a sulfate salt.
 9. The crystallinesalt form of claim 8 wherein the crystalline salt form is characterizedby an x-ray powder diffraction pattern having two or more diffractionpeaks at 2θ values selected from 9.41±0.20, 9.98±0.20, 15.17±0.20,16.70±0.20, 18.59±0.20, 19.46±0.20, 19.91±0.20, 20.63±0.20, 21.35±0.20,21.89±0.20, 23.00±0.20, 24.20±0.20, 25.40±0.20, 26.03±0.20, 27.44±0.20,28.46±0.20, 29.45±0.20, 31.22±0.20, 31.82±0.20, 33.17±0.20, 33.56±0.20,and 36.89±0.20.
 10. The crystalline salt form of claim 9 wherein thex-ray powder diffraction pattern comprises two or more diffraction peaksat 2θ values selected from 16.70±0.20, 18.59±0.20, 19.46±0.20,19.91±0.20, 23.00±0.20, and 24.20±0.20.
 11. The crystalline salt form ofclaim 8, wherein the crystalline salt form is characterized by an x-raypowder diffraction pattern in which the peak positions are substantiallyin accordance with the peak positions of the pattern shown in FIG. 4.12. The crystalline salt form of claim 8, wherein the crystalline saltform is characterized by a differential scanning calorimetry tracesubstantially in accordance with that shown in FIG.
 5. 13. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the crystalline salt form of claim 1 or claim
 2. 14. Aprocess for preparing a crystalline sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide,the process comprising: (a) contacting a protected precursor to3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide,in which the hydroxy groups are protected, with sulfuric acid to form afirst reaction mixture; (b) isolating an intermediate grade solidsulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidefrom the first reaction mixture; (c) dispersing the intermediate gradesolid sulfate salt in a diluent comprising methanol to form a secondreaction mixture; and (d) isolating the crystalline sulfate salt fromthe second reaction mixture.
 15. The process of claim 14 wherein theprotected precursor is (S)-2,2-dimethyl-[1,3]dioxolane-4-carboxylic acid{2-[3-(3-carbamoyl-phenyl)-8-azabicyclo[3.2.1]oct-8-yl]ethyl}cyclohexylmethyl-amide.16. The process of claim 15 wherein the diluent comprising methanolfurther comprises up to about 25% water.
 17. The process of claim 15wherein the diluent comprising methanol comprises between about 5% andabout 15% water.
 18. A process for preparing a crystalline sulfate saltof3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide,the process comprising: (a) contacting3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidewith sulfuric acid to form a reaction mixture; and (b) isolating thecrystalline sulfate salt from the reaction mixture.
 19. A process forpreparing a crystalline sulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide,the process comprising: (a) dispersing a crystalline hydrate of thesulfate salt of3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamidein a diluent comprising methanol to form a reaction mixture; and (b)isolating the crystalline sulfate salt from the reaction mixture. 20.The process of claim 19 wherein the diluent comprising methanol furthercomprises up to 25% water.
 21. The bisulfite adduct ofN-cyclohexylmethyl-(2-oxoethyl)-carbamic acid benzyl ester.
 22. A methodof treating a mammal having a medical condition ameliorated by treatmentwith a mu opioid receptor antagonist, the method comprisingadministering to the mammal a pharmaceutical composition comprising apharmaceutically-acceptable carrier and the crystalline salt form ofclaim 1 or claim
 2. 23. The method of claim 22 wherein the medicalcondition is opioid-induced bowel dysfunction or post-operative ileus.24. A method of enhancing motility of the gastrointestinal tract in amammal, the method comprising administering to the mammal apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and the crystalline salt form of claim 1 or claim
 2. 25. Amethod of antagonizing a mu opioid receptor in a mammal, the methodcomprising administering to the mammal a pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and the crystallinesalt form of claim 1 or claim 2.